Difference between revisions of "Part:BBa K1413002"
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<partinfo>BBa_K1413002 short</partinfo>
 
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This part consists of a sensor of phenolic compounds based on DmpR, a transcription factor of the Ntrc family.  
 
This part consists of a sensor of phenolic compounds based on DmpR, a transcription factor of the Ntrc family.  
 
<br/>Found in Pseudomonas sp. strain CF 600, DmpR regulates expression of the P0 promoter, which drives transcription of a single phenol degradation large operon  (dmpKLMNOPQBCDEFGHI). With GFP attached to the P0 promoter, it is then possible to evaluate presence of phenol by fluorescence analysis if DmpR is expressed.  
 
<br/>Found in Pseudomonas sp. strain CF 600, DmpR regulates expression of the P0 promoter, which drives transcription of a single phenol degradation large operon  (dmpKLMNOPQBCDEFGHI). With GFP attached to the P0 promoter, it is then possible to evaluate presence of phenol by fluorescence analysis if DmpR is expressed.  
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<br/>This part is basically composed of [P0 promoter + sfGFP + Pr promoter + DmpR] and allows sensing of phenolic compounds thanks to a constitutively expressed DmpR that will activate expression of GFP when bound to phenol. We characterized this biosensor using phenol as the effector.
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<br/><br/>This part is basically composed of [P0 promoter + sfGFP + Pr promoter + DmpR] and allows sensing of phenolic compounds thanks to a constitutively expressed DmpR that will activate expression of GFP when bound to phenol. We characterized this biosensor using phenol as the effector.
 
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===Usage and Biology===
 
===Usage and Biology===
 
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<br/>We prepared a protocol test to evaluate our biosensor: E.coli (DH5apha) was grown overnight in M9 medium at 37 °C and then diluted 100-fold to an OD of 0.01 in fresh M9 medium containing Chloramphenicol in 96-wells plates. After 6 hours of culture at 37 °C, each culture (200 μL) was centrifuged at 2500 r.p.m. for 15 minutes and was suspended in 200 μL of fresh M9 medium containing phenol of different concentrations. Then the fluorescence intensity of cultures was measured by microplate reader (TECAN).
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We prepared a protocol test to evaluate our biosensor: E.coli (DH5apha) was grown overnight in M9 medium at 37 °C and then diluted 100-fold to an OD of 0.01 in fresh M9 medium containing Chloramphenicol in 96-wells plates. After 6 hours of culture at 37 °C, each culture (200 μL) was centrifuged at 2500 r.p.m. for 15 minutes and was suspended in 200 μL of fresh M9 medium containing phenol of different concentrations. Then the fluorescence intensity of cultures was measured by microplate reader (TECAN).
 
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<b>Figure 1</b> describes the 96-wells plate organisation used to evaluate the biosensor.
 
<b>Figure 1</b> describes the 96-wells plate organisation used to evaluate the biosensor.
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<div align="center"><u>Figure 6 : Comparison of fluorescence intensity per cell of BBa_K1413001 (right) and BBa_K1413002 (left)</u><br/></div><br/>
 
<div align="center"><u>Figure 6 : Comparison of fluorescence intensity per cell of BBa_K1413001 (right) and BBa_K1413002 (left)</u><br/></div><br/>
  
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However this mutation makes this biosensor leakier which means that its basal fluorescence (not induced by phenol) is higher than the original one. This is responsible for the lower induction ratio observed in the case of BBa_K1413002. (Figure 7)
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However this mutation makes this biosensor leakier which means that its basal fluorescence (not induced by phenol) is higher than the original one. This is responsible for the lower induction ratio observed in the case of BBa_K1413002 <b>(Figure 7)</b>.
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<div align="center"><img alt="IMAGE" src="https://static.igem.org/mediawiki/parts/5/5d/Evry_induction_001_002.jpg" width:"100px" width="400px;" ; class="thumbimage"/></div>
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<br/> <div align="center"><u>Figure 7 : Fluorescence induction ratio of BBa_K1413001 and BBa_K1413002 </u><br/>TECAN measurement of fluorescence, 11h growth, 37 C°, M9 media (0,4% glucose). Induction ratio was obtained by dividing the fluorescence intensity of bacteria exposed to phenol by their basal fluorescence intensity (no phenol added)
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    <center><u><b>Figure 7 : Fluorescence induction ratio of BBa_K1413001 and BBa_K1413002 </b></u><br/>TECAN measurement of fluorescence, 11h growth, 37 C°, M9 media (0,4% glucose). Induction ratio was obtained by dividing the fluorescence intensity of bacteria exposed to phenol by their basal fluorescence intensity (no phenol added).</center>
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We conclude here by stating that BBaK1413002 respond with more strength to presence of phenol but is leakier making it less likely to sense small concentrations of phenol (under 1µM) than BBa_K1413001.
 
We conclude here by stating that BBaK1413002 respond with more strength to presence of phenol but is leakier making it less likely to sense small concentrations of phenol (under 1µM) than BBa_K1413001.
 
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Revision as of 21:22, 2 November 2014

P0 promoter-RBS SD001-sfGFP- Terminator B0015 - Pr promoter-DmpR

This part consists of a sensor of phenolic compounds based on DmpR, a transcription factor of the Ntrc family.
Found in Pseudomonas sp. strain CF 600, DmpR regulates expression of the P0 promoter, which drives transcription of a single phenol degradation large operon (dmpKLMNOPQBCDEFGHI). With GFP attached to the P0 promoter, it is then possible to evaluate presence of phenol by fluorescence analysis if DmpR is expressed.

This part is basically composed of [P0 promoter + sfGFP + Pr promoter + DmpR] and allows sensing of phenolic compounds thanks to a constitutively expressed DmpR that will activate expression of GFP when bound to phenol. We characterized this biosensor using phenol as the effector.

Usage and Biology

We prepared a protocol test to evaluate our biosensor: E.coli (DH5apha) was grown overnight in M9 medium at 37 °C and then diluted 100-fold to an OD of 0.01 in fresh M9 medium containing Chloramphenicol in 96-wells plates. After 6 hours of culture at 37 °C, each culture (200 μL) was centrifuged at 2500 r.p.m. for 15 minutes and was suspended in 200 μL of fresh M9 medium containing phenol of different concentrations. Then the fluorescence intensity of cultures was measured by microplate reader (TECAN).

Figure 1 describes the 96-wells plate organisation used to evaluate the biosensor. We used three control in this experiment :

  • Media only : to evaluate the natural fluorescence of the media with different concentrations of phenol.
  • pSB1C3 : DH5alpha resistant to chloramphenicol used as a growth control.
  • BBa_J23106 : DH5alpha carrying BBa J23106, allowing constitutive production of GFP. This control was used to evaluate the eventual impact of phenol on GFP expression and/or fluorescence.
Purified GFP : Used to associated fluorescence values to a defined concentration of GFP.

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Figure 1 : 96-wells plate organisation scheme


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Figure 2 : OD600 values measured over 11h in 96-wells plate(TECAN)


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Figure 3 : Fluorescence intensity per cell of BBa_K1413002
TECAN measurement of fluorescence during 11h growth, 37 C°, M9 media (0,4% glucose). The values were obtained by substracting raw Fluorescence values of bacteria exposed to phenol by fluorescence of media (M9) then dividing by corresponding OD600.

These data show an increase in expression of sfGFP in response to increasing concentration of phenol present in wells.They also show that the biosensor is able to sense down to 1µM of phenol. The induction ratio calculated from these data (Figure 4) shows a 3-fold increase of fluorescence at 1µM of phenol and up to 37-fold increase at 1000µM.
IMAGE
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Figure 4 : Fluorescence induction ratio of BBa_K1413002
TECAN measurement of fluorescence, 11h growth, 37 C°, M9 media (0,4% glucose). Induction ratio was obtained by dividing the fluorescence intensity of bacteria exposed to phenol by their basal fluorescence intensity (no phenol added)


IMAGE
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Figure 5 : Fluorescence intensity per cell of BBa_K1413001 and BBa_K1413002
TECAN measurement of fluorescence during 11h growth, 37 C°, M9 media (0,4% glucose). The values were obtained by substracting raw Fluorescence values of bacteria exposed to phenol by fluorescence of media (M9) then dividing by corresponding OD600.


Comparison of BBa_K1413001 vs BBa_K1413002 (new RBS).
We decided to strenghten the signal produced by our biosensor by mutating the ribosome binding site of sfGFP. This mutation is processed in a way that it reproduce the consensus sequence of Shine Dalgarno (AGGAGGUAA)allowing mRNA to bind more specifically to 16s rRNA. This in turn increases the translation rate of the mRNA. See design page.
Comparison of fluorescence per cell reveals that BBa_K1413002 produces significantly more fluorescence than BBa_K1413001 from 10µM to 1000µM of phenol.The data also show that by mutating the RBS, fluorescence is produced more rapidly.

IMAGEIMAGE
Figure 6 : Comparison of fluorescence intensity per cell of BBa_K1413001 (right) and BBa_K1413002 (left)

However this mutation makes this biosensor leakier which means that its basal fluorescence (not induced by phenol) is higher than the original one. This is responsible for the lower induction ratio observed in the case of BBa_K1413002 (Figure 7).

IMAGE
Symbol
Figure 7 : Fluorescence induction ratio of BBa_K1413001 and BBa_K1413002
TECAN measurement of fluorescence, 11h growth, 37 C°, M9 media (0,4% glucose). Induction ratio was obtained by dividing the fluorescence intensity of bacteria exposed to phenol by their basal fluorescence intensity (no phenol added).

We conclude here by stating that BBaK1413002 respond with more strength to presence of phenol but is leakier making it less likely to sense small concentrations of phenol (under 1µM) than BBa_K1413001.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1241
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1719
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1404
    Illegal BsaI.rc site found at 1945
    Illegal SapI.rc site found at 211
    Illegal SapI.rc site found at 2602